The success of malaria eradication efforts hinges on the development of a safe and efficacious malaria vaccine that induces complete protection against infection. To date, efforts to develop such a vaccine for worldwide use have been unsuccessful. However, vaccination with the subunit RTS,S/AS02 vaccine encoding the Plasmodium falciparum circumsporozoite protein significantly reduced deaths due to severe malaria in Africa children and demonstrated for the first time that development of an anti-infection malaria subunit vaccine is feasible. We know that live-attenuated Plasmodium parasites yield complete and long-lasting CD8+ cytotoxic T lymphocyte (CTL)-mediated protection in mice and humans, but such vaccine preparations are not easily deployed in the field. The dependence of protection on antigens targeted by CTLs indicates that the targets of such cells would make ideal subunit vaccine antigens. The search for such targets was previously impossible on a whole organism scale because attenuated parasite vaccines contain thousands of different immune targets leading to complex polyspecific CTL responses - until now there was no methodology capable of identifying discrete antigenic targets in the midst of such a complex response. To solve this problem, I will employ a high-throughput method to efficiently screen thousands of candidate targets against CTLs from malaria-exposed mice in order to decipher the key CTL responses that confer immunity against malaria. I hypothesize that protective CTL responses encompass a wide range of antigenic specificities and include parasite proteins expressed in both liver and erythrocyte stages. These target antigens can be rapidly identified using this high-throughput minigene-driven screening approach. In Specific Aim 1, I will combine this approach with different vaccination models in mice to assess the kinetics and diversity of CTLs and identify cognate target antigens associated with protection. I will also use complementary experimental and in silico approaches to guide development of a focused candidate target library. In Specific Aim 2, I will evaluate cross-priming of CTLs by malaria-infected erythrocytes and determine how such targets elicit cross-stage CTL immunity. Immune targets defined in this work can later be used to rationally test CTLs from malaria-exposed humans to define a polyspecific protective CTL repertoire in humans. The proposed approach will serve as a powerful paradigm for identifying protective CTL targets and can be generally applied to malaria and other infections. This application addresses PA-10-059 (K08 Career Development Award) and was crafted to ensure excellent mentorship, strong institutional support and successful collaborations. NARRATIVE Global eradication of malaria requires an efficacious anti-infection malaria vaccine. Development of such a vaccine requires the identification of discrete antigenic targets from among thousands of proteins expressed by malaria parasites. We propose here to study CTLs induced by different immunization approaches in mice and identify protective antigens by profiling CTL responses using high-throughput screening technologies and other cutting-edge approaches to accelerate malaria vaccine development.